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Contribution of the active site aspartic acid to catalysis in the bacterial neuraminidase from Micromonospora viridifaciens
Author(s) -
Watson Jacqueline N.,
Newstead Simon,
Dookhun Veedeeta,
Taylor Garry,
Bennet Andrew J.
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2004.10.016
Subject(s) - aspartic acid , neuraminidase , active site , sialidase , biochemistry , hydrolysis , residue (chemistry) , chemistry , mutant , glycine , carboxylate , stereochemistry , recombinant dna , enzyme , biology , amino acid , gene
A recombinant D92G mutant sialidase from Micromonospora viridifaciens has been cloned, expressed and purified. Kinetic studies reveal that the replacement of the conserved aspartic acid with glycine results in a catalytically competent retaining sialidase that possesses significant activity against activated substrates. The contribution of this aspartate residue to the free energy of hydrolysis for natural substrates is greater than 19 kJ/mol. The three dimensional structure of the D92G mutant shows that the removal of aspartic acid 92 causes no significant re‐arrangement of the active site, and that an ordered water molecule substitutes for the carboxylate group of D92.